Peptides for gene delivery

ABSTRACT

Compositions and methods are described for nucleic acid formulation for gene delivery. A new class of low molecular weight condensing agents, namely aromatic amino acid—containing peptides, are described for use in receptor-mediated and nonreceptor-mediated gene delivery, both in vivo and in vitro.

[0001] This application is a continuation-in-part application of U.S.patent application Ser. No. 08/743,269, filed Nov. 4, 1996.

[0002] This invention was made with government support awarded by theNational Institutes of Health (grant number GM48049). The government hascertain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates to the introduction of genes intocells. In particular, the present invention relates to compositions andmethods of nucleic acid formulation for gene delivery.

BACKGROUND

[0004] There are a number of techniques for the introduction of genesinto cells. One common method involves viruses that have foreign genes(e.g., transgenes) incorporated within the viral DNA. However, the viralgenes are also delivered with the desired gene and this can lead toundesirable results.

[0005] Nonviral gene delivery systems are being developed to transfectmammalian host cells with foreign genes. In such approaches, nucleicacid is typically complexed with carriers that facilitate the transferof the DNA across the cell membrane for delivery to the nucleus. Theefficiency of gene transfer into cells directly influences the resultantgene expression levels.

[0006] The carrier molecules bind and condense DNA into small particleswhich facilitate DNA entry into cells through endocytosis orpinocytosis. In addition, the carrier molecules act as scaffolding towhich ligands may be attached in order to achieve site specifictargeting of DNA.

[0007] The most commonly used DNA condensing agent for the developmentof nonviral gene delivery systems is polylysine in the size range of dp90-450. Its amino groups have been derivatized with transferrin,glycoconjugates, folate, lectins, antibodies or other proteins toprovide specificity in cell recognition, without compromising itsbinding affinity for DNA. However, the high molecular weight andpolydispersity of polylysine also contribute to a lack of chemicalcontrol in coupling macromolecular ligands which leads to heterogeneityin polylysine-based carrier molecules. This can complicate theformulation of DNA carrier complexes and limits the ability tosystematically optimize carrier design to achieve maximal efficiency.

[0008] Clearly, there is a need for improved methods of gene delivery.Such methods should be amenable to use with virtually any gene ofinterest and permit the introduction of genetic material into a varietyof cells and tissues.

SUMMARY OF THE INVENTION

[0009] The present invention relates to the introduction of genes intocells. In particular, the present invention relates to compositions andmethods of nucleic acid formulation for gene delivery. The inventioncontemplates cationic peptides containing aromatic amino acids (i.e.,phenylalanine, tyrosine and tryptophan) and in particular,tryptophan-containing peptides that mediate gene transfer by condensingDNA into small particles.

[0010] The present invention contemplates methods for introducingnucleic acid into cells (both in vivo and in vitro). In one embodiment,the method comprises a) providing: i) an aromatic amino acid—containingpeptide capable of binding to nucleic acid, ii) nucleic acid encodingone or more gene products, and iii) cells capable of receiving saidnucleic acid, said cells having cell membranes; b) binding said peptideto said nucleic acid to make a complex; c) introducing said complex tosaid cells under conditions such that said complex is delivered acrosssaid cell membrane.

[0011] While it is not intended that the invention be limited by thelength of the peptide, it is preferred that the peptides of the presentinvention are less than forty amino acids in length, more preferablyless than thirty amino acids in length, and most preferably, less thantwenty amino acids in length.

[0012] It is also not intended that the present invention be limited bythe precise composition of the peptides. A variety of peptidescontaining aromatic amino acids are contemplated. In one embodiment, thepeptides of the present invention comprise L-lysine (Lys) and tryptophan(Trp). In another embodiment,the peptides of the present inventioncontain L-lysine (Lys), tryptophan (Trp) and cysteine (Cys). In apreferred embodiment, a peptide is contemplated that demonstrates highactivity in mediating gene transfer in cell culture, said peptide havingthe structure: Cys-Trp-(Lys)₁₈. Other peptides (including peptides withtwo, three and four tryptophan residues) are contemplated.

[0013] The present invention also contemplates the use of the peptidesof the present invention in receptor-mediated gene transfer (both invitro and in vivo). In one embodiment, the method comprises linking theDNA to a cationic peptide of the present invention (usually an aromaticamino acid—substituted poly-L-lysine) containing a covalently attachedligand, which is selected to target a specific receptor on the surfaceof the tissue of interest. The gene is taken up by the tissue,transported to the nucleus of the cell and expressed for varying times.

[0014] In one embodiment, the receptor-mediated method of the presentinvention for delivering an oligonucleotide to cells of an animal,comprises a) providing: i) a target binding moiety capable of binding toa receptor present on the surface of a cell in a tissue of an animal,ii) an aromatic amino acid—substituted polylysine capable of binding tonucleic acid, iii) an oligonucleotide encoding one or more geneproducts, and iv) a recipient animal having cells, said cells havingsaid receptor; b) conjugating said target binding moiety to saidsubstituted polylysine to form a carrier; c) coupling said carrier withsaid oligonucleotide to form a pharmaceutical composition; and d)administering said composition to said recipient animal under conditionssuch that said oligonucleotide is delivered to said cells.

[0015] As noted above, the present invention contemplates polylysinepeptides containing tryptophan for use in gene delivery. In oneembodiment, the synthetic peptides contemplated possess a lysine repeatvarying from between 3 and 36 residues and comprise one or moretryptophan and cysteine residues. In a preferred embodiment, the peptidecomprises 13-18 lysine residues; such peptides which possess a singletryptophan residue enhances gene transfer to cells in culture by up tothree orders of magnitude relative to comparable polylysine peptidelacking a tryptophan.

[0016] An understanding of how the peptides of the present inventionimprove the gene delivery in a superior manner is not required topractice the present invention. Nonetheless, it is believed that themechanism of peptide mediated gene transfer is related to the efficiencyof condensing DNA into small particles. While not limited to anyparticular theory, it is believed that tryptophan plays a specific rolein organizing the DNA binding of cationic peptide to produce smallcondensates that exhibit enhanced gene transfer efficiency. In thismanner, the tryptophan-containing peptides of the present inventionrepresent a new class of low molecular weight condensing agents that maybe modified with ligands to produce low molecular weight carriers forsite specific gene delivery.

[0017] It is not intended that the present invention be limited by thenature of the nucleic acid. The target nucleic acid may be native orsynthesized nucleic acid. The nucleic acid may be from a viral,bacterial, animal or plant source.

DEFINITIONS

[0018] To racilitate understanding of the invention, a number of termsare defined below.

[0019] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity is retained.

[0020] The term “wild-type” refers to a gene or gene product which hasthe characteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product which displaysmodifications in sequence and or functional properties (i.e., alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these areidentified by the fact that they have altered characteristics whencompared to the wild-type gene or gene product.

[0021] The term “oligonucleotide” as used herein is defined as amolecule comprised of two or more deoxyribonucleotides orribonucleotides, usually more than three (3), and typically more thanten (10) and up to one hundred (100) or more. The exact size will dependon many factors, which in turn depends on the ultimate function or useof the oligonucleotide. The oligonucleotide may be generated in anymanner, including chemical synthesis, DNA replication, reversetranscription, or a combination thereof.

[0022] Because mononucleotides are reacted to make oligonucleotides in amanner such that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an end of an oligonucleotide is referred to asthe “5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends.

[0023] The term “label” as used herein refers to any atom or moleculewhich can be used to provide a detectable (preferably quantifiable)signal, and which can be attached to a nucleic acid or protein. Labelsmay provide signals detectable by fluorescence, radioactivity,colorimetry, gravimetry, X-ray diffraction or absorption, magnetism,enzymatic activity. and the like.

[0024] The terms “nucleic acid substrate” and nucleic acid template” areused herein interchangeably and refer to a nucleic acid molecule whichmay comprise single- or double-stranded DNA or RNA.

[0025] The term “substantially single-stranded” when used in referenceto a nucleic acid substrate means that the substrate molecule existsprimarily as a single strand of nucleic acid in contrast to adouble-stranded substrate which exists as two strands of nucleic acidwhich are held together by inter-strand base pairing interactions.

[0026] The term “sequence variation” as used herein refers todifferences in nucleic acid sequence between two nucleic acid templates.For example, a wild-type structural gene and a mutant form of thiswild-type structural gene may vary in sequence by the presence of singlebase substitutions and/or deletions or insertions of one or morenucleotides. These two forms of the structural gene are said to vary insequence from one another. A second mutant form of the structural genemay exist. This second mutant form is said to vary in sequence from boththe wild-type gene and the first mutant form of the gene. It is noted,however, that the invention does not require that a comparison be madebetween one or more forms of a gene to detect sequence variations.

[0027] The “target cells” may belong to tissues (including organs) ofthe organism, including cells belonging to (in the case of an animal)its nervous system (e.g., the brain, spinal cord and peripheral nervouscells), the circulatory system (e.g., the heart, vascular tissue and redand white blood cells), the digestive system (e.g., the stomach andintestines), the respiratory system (e.g., the nose and the lungs), thereproductive system, the endocrine system (the liver, spleen, thyroids,parathyroids), the skin, the muscles, or the connective tissue.

[0028] Alternatively, the cells may be cancer cells derived from anyorgan or tissue of the target organism, or cells of a parasite orpathogen infecting the organism, or virally infected cells of theorganism.

[0029] Exogenous DNA has been introduced into hepatocytes by targetingthe asialoglycoprotein (ASGP) receptor by means of aligand-poly-L-lysine bioconjugate. See U.S. Pat. No. 5,166,320, herebyincorporated by reference. Such receptor-mediated approaches can be usedin combination with the novel peptides of the present invention.

[0030] Exogenous DNA has also been introduced for antisense treatment.See U.S. patent appl. Ser. No. 08/042,943, filed Apr. 5, 1993 andcorresponding PCT Publication No. WO 94/23050, hereby incorporated byreference. Such antisense approaches can be used in combination with thenovel peptides of the present invention.

DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 graphically depicts light scattering results for peptidesof the present invention (each titration represents the average of threedeterminations).

[0032]FIG. 2 graphically shows the percent of DNA sedimented followingcentrifugation of peptide induced DNA condensates.

[0033]FIG. 3 shows reporter gene expression for DNA condensates. Panel Adepicts the results for HepG2 cells while panel B shows the results ofCOS 7 cells.

[0034]FIG. 4 is a dose response curve showing gene expression in HepG2cells.

DESCRIPTION OF THE INVENTION

[0035] The present invention relates to the introduction of genes intocells. In particular, the present invention relates to compositions andmethods of nucleic acid formulation for gene delivery. The aromaticamino acid—containing peptides of the present invention represent a newclass of low molecular weight condensing agents for gene delivery.

[0036] Cationic peptides possessing a single cysteine, tryptophan and alysine repeat were synthesized to define the minimal peptide lengthneeded to mediate transient gene expression in mammalian cells. TheN-terminal cysteine in each peptide was either alkylated or oxidativelydimerized to produce peptide possessing lysine chains of 3, 6, 8, 13,16, 18, 26, and 36 residues. Each synthetic peptide was studied for itsability to condense plasmid DNA and compared to polylysine₁₉ andcationic lipids to establish relative in vitro gene transfer efficiencyin HepG2 and COS 7 cells.

[0037] Peptides with lysine repeats of 13 or more bound DNA tightly andproduced condensates that decreased in mean diameter from 231 nm down to53 nm as the lysine chain length increased. In contrast, peptidespossessing 8 or fewer lysine residues were similarly to polylysine₁₉which bound DNA weakly and produced large (0.7-3 μm) DNA condensates.

[0038] The luciferase expression was elevated a thousand-fold aftertransfecting HepG2 cells with DNA condensates prepared with alkylatedCys-Trp-Lys₁₈ and cationic lipids were equivalent in HepG2 cells butdifferent by ten-fold in COS 7 cells.

[0039] A forty-fold reduction in particle size and a thousand-foldamplification in transfection efficiency for AlkCWK₁₈ DNA condensatesrelative to polylysine₁₉ DNA condensates suggests a contribution fromtryptophan that leads to enhanced gene transfer properties for AlkCWK₁₈Tryptophan containing cationic peptide result in the formation of smallDNA condensates that mediate efficient nonspecific gene transfer inmammalian cells. Due to their low toxicity, these peptide may findutility as carriers for nonspecific gene delivery or may be developedfurther as low molecular weight DNA condensing agents used in targetedgene delivery systems.

EXPERIMENTAL

[0040] The following examples serve to illustrate certain preferredembodiments and aspects of the present invention and are not to beconstrued as limiting the scope thereof.

[0041] In the experimental disclosure which follows, the followingabbreviations apply: ° C. (Centigrade); μg (micrograms); μmole(micromoles); μl (microliters); mL (milliliters); mM (milliMolar);RP-HPLC (reverse phase high performance liquid chromatography); CWK(cysteine-tryptophane-lysine; TFA (trifluoroacetic acid); EDF(ethanedithiol); MALDI-TOF-MS (matrix assisted time of flight massspectrometry); RLU (relative light units); DTT (dithiothreitol); FBS(fetal bovine serum); MEM (minimal essential media); DMEM (Dulbecco'smodified Eagel media); HBM (Hepes buffered mannitol); QELS (quasielastic light scattering).

[0042] In the examples described below, N-terminal Fmoc protected aminoacids, and all other reagents for peptide synthesis were obtained fromAdvanced ChemTech (Lexington, Ky.). Minimum essential media (MEM),Sephadex G25, dithiothreitol, iodoacetamide, iodoacetic acid andpolylysine₁₉ (MW 1000-4000) were purchased from Sigma Chemicals (St.Louis, Mo.). Ethanedithiol (EDT) was purchased from Aldrich Chemical(Milwaukee, Wis.). Trifluoroacetic acid (TFA) was purchased from FisherScientific (Pittsburgh, Pa.). LB media, LB agar, D-luciferin, andluciferase from Photinus pyralis (EC 1.13.12.7) were obtained fromBoehringer Mannheim (Indianapolis, Ind.). HepG2 and COS 7 cells werefrom the American Type Culture Collection (Rockville, Md.). Dulbecco'smodified Eagle medium (DMEM), media supplements and heat inactivated“qualified” fetal bovine serum (FBS) were from Gibco BRL (Grand Island,N.Y.). Bradford reagent was purchased from BioRad (Hercules, Calif.) andthiazole orange was a gift from Beckton Dickinson ImmunocytometrySystems (San Jose, Calif.). The 5.6 kbp plasmid pCMVL encoding thereporter gene luciferase under the control of the cytomegaloviruspromoter was a gift from Dr. M. A. Hickman at the University ofCalifornia, Davis. Peptide purification was performed using asemi-preparative (10 μm) C18 RP-HPLC column from Vydac (Hesperia,Calif.). HPLC was performed using a computer interfaced HPLC andfraction collector from ISCO (Lincoln, Nebr.).

[0043] Plasmid DNA was prepared by the alkaline lysis method andpurified on cesium chloride gradient. Peptide were prepared by solidphase peptide synthesis on Fmoc-L-Boc-lysine-Wang resin(p-benzyloxybenzyl alcohol resin, 1% divinyl benzene cross linked100-200 mesh) at a 136 μmol scale (0.68 mmol/g resin).

[0044] Peptide DNA condensates were prepared at a final DNAconcentration of 20 μg/ml and at a peptide/DNA stoichiometry varyingfrom 0.1 to 1.5 nmol of peptide per μg of DNA. The condensates wereformed by adding peptide (2-30 nmol) prepared in 500 μl of isotonicHepes buffered mannitol (HBS, 0.27 M mannitol, 5 mM sodium Hepes, pH7.5) to 20 μg of DNA in 500 μl HBM while vortexing, followed byequilibration at room temperature for 30 min.

[0045] Sedimentation of DNA condensates was evaluated by measuring theconcentration of DNA in solution before and after centrifugation. Afterforming peptide DNA condensates as described above, a 50 μl aliquot (1μg of DNA) was diluted in 1 ml of water and the AbS_(260nm) wasdetermined on a Beckman DU640 spectrophotometer. Followingcentrifugation at 13,000 g for 4 min at room temperature an identicalaliquot was diluted with 1 ml of water and the concentration of DNAremaining in solution was determined. The ratio of absorbancessubtracted from unity and multiplied by 100 was defined as the percentsedimentation.

[0046] Peptide binding to DNA was monitored by a fluorescence titrationassay. A 1 μg aliquot of the peptide DNA condensate prepared asdescribed above was diluted to 1 ml in HBM containing 0.1 μM thiazoleorange. The fluorescence of the intercalated dye was measured on anLS50B fluorometer (Perkin Elmer, UK) in a micro cuvette by exciting at500nm while monitoring emission at 530 nm, with the slits set at 15 and20 nm and photomultiplier gain set to 700 volts. DNA condensation wasmonitored by measuring total scattered light at 90° by setting bothmonochromators to 500 nm and decreasing slit widths to 2.5 nm.Fluorescence and scattered light intensity blanks were subtracted fromall values before data analysis.

[0047] Transmission electron microscopy was preformed by immobilizingcondensed DNA on carbon coated copper grids (3 mm diameter, 400 mesh;Electron Microscopy Sciences, Fort Washington, Pa.). Grids were glowdischarged and 3 μl of peptide DNA condensate (20 μg/ml), prepared asdescribed above, was placed on the grid for 5 min. The grids wereblotted dry then stained by floating for 1.5 min on each of three 100 μldrops of urinal acetate (1%, in 95% ethanol) followed by rinsing with0.4% detergent solution (PhotoFlo, Kodak), and drying. Electronmicroscopy was performed using a Philips EM-100 transmission electronmicroscope.

[0048] Particle size analysis was measured for peptide DNA condensatesprepared at a DNA concentration of 20 μg of DNA. Samples were analyzedusing a Nicomp 370 Autodilute submicron particle sizer in the solidparticle mode and acquisition was continued until the fit error was lessthan ten. The mean diameter and population distribution were computedfrom the diffusion coefficient using functions supplied by theinstrument.

EXAMPLE 1

[0049] Cationic peptides were designed to probe the minimal size neededto mediate efficient gene transfer in mammalian cells. The syntheticstrategy involved comparison of four peptides with varying.lysine chainlength in the range of 3-18 residues. During peptide synthesis,truncated peptides were capped by N-acetylation and a tryptophan residuewas placed near the N-terminus to provide a chromophore foridentification of full length sequences during purification. Thisresidue allows quantitation of peptide concentration and is alsointended for use in monitoring fluorescence to evaluate peptide bindingto DNA as previously described. In addition, each peptide possessed anN-terminal cysteine residue as a potential ligand attachment site.

[0050] The synthesis was accomplished using a computer interfaced Model90 synthesizer from Advanced Chemtech, Lexington, Ky. Lysine andtryptophan side chains were Boc protected and the sulfhydryl side chainof cysteine was protected with a trityl group. A six molar excess ofN-terminal Fmoc protected amino acid was activated in situ in thereaction vessel by adding equimolar diisopropylcarbodiimide andN-hydroxybenzotriazole in a total reaction volume of 18 ml. Coupling wascarried out for 1 hr and was followed with a capping cycle for 30 minwith 10% acetic anhydride in 1% diisopropylethylamine. Fmoc deblockingwas performed with 25% piperidine for 12 min. All reagents weredissolved in dimethyl formamide.

[0051] At completion, the resin conjugated peptide was washed withdichloromethane, dried and weighed. Cleavage was performed in a solutionof TFA:EDT:water (95:2.5:2.5 v/v) for 30 min at room temperature, whichsimultaneously deprotected the amino acid side chains. The peptidesolution was extracted with diethyl ether, concentrated by rotaryevaporation,and freeze dried. Lyophilized crude peptide were dissolvedin degassed and nitrogen purged 0.1% TFA. peptide (3 μmol per injection)were purified on a semi-preparative (2×25 cm) C18 RP-HPLC column elutedat 10 ml/min with 0.1% TFA and acetonitrile (5-20% over 40 min) whilemonitoring absorbance at 280 nm, 1.0 AUFS. Purified peptide wereconcentrated by rotary evaporation, lyophilized, and stored dry at −20°C.

[0052] Lyophilized peptide (1 μmol) were dissolved in 1 ml of nitrogenpurged 50 mM Tris hydrochloride (pH 7.5) and reduced by the addition of250 μl of 100 mM dithiothreitol

was carried out by adding 25 mg of solid iodoacetamide or iodoaceticacid followed by reacting for 1 hour at room temperature. The alkylatedpeptide were acidified to pH 2.0 with TFA and purified by RP-HPLC asdescribed above. The yield of each purified peptide (approx. 25%) wasdetermined from the absorbance of tryptophan (ε_(280nm)=5600 M⁻¹cm⁻¹).The TFA salt of polylysine₁₉ was prepared by chromatographing thehydrobromide salt on RP-HPLC eluted with 0.1% TFA and acetonitrile whiledetecting 214 nm as described above. The concentration of polylysine₁₉was established by fluorescamine analysis using a calibrated standard ofAlkCWK₁₈ as a reference.

[0053] Dimeric peptides were prepared by dissolving 1 μmol of eachpurified CWK_(n) (n=3,8,13, or 18) peptide in Tris hydrochloride pH 7.5followed by reaction at 37° C. for 24 hrs. Each dimeric peptide waspurified using RP-HPLC as described above and quantified by AbS_(280nm)(ε=11,200 M⁻¹cm⁻¹).

[0054] Peptides were alkylated with iodacetamide to provide AlkCWK_(n)(where n=3, 8, 13 or 18 residues) (Table I). A further extension of thispeptide series was accomplished by allowing the cysteine of eachmonomeric peptide to oxidize, resulting in a panel of homodimericpeptides each possessing two tryptophans and a discontinuous lysinerepeat of TABLE I Peptides For Gene Delivery Name Sequence Mass(Obs./Calc^(a)) AlkCWK₃ Alk-S-Cys-Trp-(Lys)₃ 750.2/750.0 AlkCWK₈Alk-S-Cys-Trp-(Lys)₈ 1391.1/1390.9 AlkCWK₁₃ Alk-S-Cys-Trp-(Lys)₁₃2031.1/2031.8 AlkCWK₁₈ Alk-S-Cys-Trp-(Lys)₁₈ 2672.7/2672.5 DiCWK₃(Lys)₃-Trp-Cys-S-S-Cys-Trp-(Lys)₃ 1382.5/1382.8

DiCWK₁₃ (Lys)₁₃-Trp-Cys-S-S-Cys-Trp-(Lys)₁₃ 3946.2/3945.9 DiCWK₁₈(Lys)₁₈-Trp-Cys-S-S-Cys-Trp-(Lys)₁₈ 5227.8/5227.9 Polylysine₁₉ (Lys)₁₉   n.d^(b)/2435.8

[0055] either 6, 15, 26, or 36 residues in length (Table I).Substitution of iodacetic acid for iodacetamide in the alkylation stepled to an AlkCWK₁₈ peptide that was acid stable and was functionallyequivalent in formulation and biological assays.

[0056] Peptide were characterized using MALDI-TOF-MS which produced adominant ion corresponding to the anticipated molecular weight of eachpeptide (Table I).The peptide (1 nmol) was reconstituted in 100 μl of0.1% acetic acid and 1 μl was applied to the target and analyzed using aVestec-2000 internal standard. The instrument was operated with 23 KVion accelerating voltage and a 3 KV multiplier voltage using a 337 nmVSL-SS&ND nitrogen laser with a 3 ns pulse width.

[0057] Peptides were studied for DNA binding using a dye exclusionassay. Peptide binding to DNA leads to exclusion of thiazole orangeintercalation and a decrease in fluorescence.

peptide per μg of DNA led to a reduction in fluorescence except for thesmallest peptide (AlkCWK₃) which failed to exclude the intercalatorwithin the titration range (FIG. 1A). An asymptote in the fluorescencedecline was observed at a stoichiometry of 0.6, o.4 or 0.2 nmol ofpeptide per μg of DNA for AlkCWK_(8,13, or 18), respectively (FIG. 1A).The relative fluorescence intensity at peptide/DNA stoichiometries abovethe asymptote established that AlkCWK_(13 and 18) were able to excludethiazole orange intercalation more efficiently than AlkCWK₈.

[0058] Dimeric peptides (DiCWK_(n) n=8, 13, 18) also possessed highaffinity for DNA as evidenced by the stoichiometry of the fluorescenceasymptote and the reduction is residual fluorescence, both of whichcorrelated with the number of lysine residues (FIG. 1B). Of this series,DiCWK₃ possessed weak affinity for DNA and thereby produced an asymptoteat a stoichiometry of 1 nmol of peptide per μg of DNA.

[0059] In contrast to these results, polylysine₁₉ demonstrated amarkedly different fluorescence titration curve compared to thealkylated or dimeric peptides of comparable length (FIG. 1C). Eventhough polylysine₁₉ has a similar number of lysine residues as AlkCWK₁₈its fluorescence asymptote occurs at a stoichiometry of approximately0.6 nmol of peptide per μg of DNA. This result suggests thatpolylysine₁₉ binding to DNA is weak relative to AlkCWK₁₈.

[0060] Total light scattering at 90° was used to detect the peptidestoichiometry at which condensed DNA particles were formed. Titration ofeither AlkCWK_(8, 13, or 18) with DNA produced a maximal total lightscattering at stoichiometries that corresponded to the asymptoteobserved in the fluorescence exclusion assay (FIG. 1D). A plateau in thelight scattering profile observed at a stoichiometry of 0.6, 0.4, and0.2 for AlkCWK_(8, 13, or 18), respectively. established the completecondensations of DNA at or above this peptide/DNA ratio. In contrast,titration of DNA with AlkCWK₃ failed to produce an increase in the lightscattering, supporting earlier observations that indicate AlkCWK₃ failsto bind to DNA.

[0061] Titration of the dimeric peptides with DNA each producedcondensates detected by light scattering (FIG. 1E). Although the plateaulight scattering level for each dimeric peptide DNA condensate wasnearly indistinguishable, the stoichiometry at which the plateau was

respectively. A weaker binding affinity for DiCWK₃ was evident from theplateau in light scattering which occurred at a stoichiometry of 1 nmolper μg of DNA (FIG. 1E).

[0062] The light scattering profile for polylysine₉ was very distinctfrom that obtained for alkylated and dimeric peptides. A sharp increaseoccurred at a stoichiometry of 0.4 nmol per μg of DNA which declined toapproximately 50 light scattering units at higher peptide/DNAstoichiometries (FIG. 1F). This light scattering titration profiledistinguished the binding properties of polylysine₁₉ from CWK₁₃peptides, suggesting differences in the particle size for potylysine₁₉DNA condensates.

[0063] To evaluate the relative particle size of DNA condensatesprepared at stoichiometries ranging from 0.1-1.5 nmol of peptide per μgof DNA, a sedimentation assay was utilized to measure the DNA remainingin suspension following centrifugation at 13,000×g for 4 min 18) (FIG.2). Titration of DNA with AlkCWK₃ resulted in the complete recovery ofthe DNA following centrifugation, supporting earlier findings thatindicate AlkCWK₃ fails to bind and condense DNA into particles.Alternatively, AlkCWK_(8, 13, and 18) each produced maximalsedimentation at a stoichiometry which roughly correlates with thestoichiometry calculated for a charge neutral complex (FIG. 2A). Atstoichiometries greater than charge neutral, AlkCWK₈ condensatessedimented to a greater extent than AlkCWK₁₃ or AlkCWK₁₈ condensates,indicating their larger size.

[0064] A similar trend was observed when sedimenting dimeric peptide DNAcondensates. The maximal sedimentation was observed at a stoichiometryof 0.8, 0.2, 0.15 and 0.1 nmol of peptide per μg of DNA forDiCWK_(3, 8, 13, and 18), respectively, (FIG. 2B). At stoichiometriesabove the calculated charge neutral point DiCWK_(8, 13, and 18) DNAcondensates failed to sediment suggesting they are smaller in size (FIG.2B). It is also evident that DiCWK₃ DNA condensates were large due tothe observed sedimentation (70-80%) at stoichiometries above the chargeneutralization point (FIG. 2B).

[0065] In contrast, polylysine₁₉ DNA condensates sedimented completelyat 0.2 nmol of peptide per μg of DNA and failed to recover at higherstoichiometries. These data established that once polylysine₉ DNAcondensates are formed, they remained large throughout the titrationrange (FIG. 2C).

[0066]

polylysine₁₉ at a stoichiometry of 0.8 nmol of peptide per μg of DNA andparticle sizes were compared using quasi elastic light scattering(QELS). A population of particles with average diameters of 0.7-3.1 μmwas determined for polylysine₁₉ AlkCWK₃ and DiCWK₃ DNA condensateswhereas no particles were detected for AlkCWK₃ DNA condensates (TableII), consistent with the results of sedimentation analysis.

[0067] Each alkylated or dimeric peptide possessing thirteen lysineresidues or more produced a population of particles with mean diametersof 53-231 nm (Table II). It should be noted that particle populationswere most often bimodal, possessing a major (>90%) smaller diameterpopulation and a minor larger diameter population which contributed tothe large standard deviation of the average particle size (Table II).

[0068] Particle sizes determined by QELS were substantiated by analyzingDNA condensates using electron microscopy. The images (not shown)demonstrate that condensates produced with AlkCWK₁₈ are relativelyuniform particles with diameters of approximately 50-100 nm whereaspolylysine₁₉ induced condensates were large flocculated particles,consistent with the result of particle size analysis by QELS. TABLE IIQELS Particle Size Distribution Particle Size Population Peptide DNACondensate^(a) Diameter^(b) (nm)

(nm)^(c) Polylysine₁₉ 3102 297 A1kCWK₃ — — AlkCWK₈ 2412 354

A1kCWK₁₈ 78 30 DiCWK₃ 724 154 DiCWK₈ 53 24 DiCWK₁₃ 56 29 DiCWK₁₈ 64 27

EXAMPLE 2 In Vitro Gene Transfection

[0069] HepG2 cells (2×10⁶ cells) were plated on 6×35 mm wells and grownto 40-70% confluency in MEM supplemented with 10% FBS, penicillin andstreptomycin (10,000 U/ml), sodium pyruvate (100 mM), and L-glutamine(200 mM). Transfections were performed in MEM (2 ml per 35 mm well) with2% FBS, with or without 80 μM chloroquine. Peptide DNA condensates (10μg of DNA in 0.5 ml HBM) were added drop wise to triplicate wells. After5 h incubation at 37° C., the media was replaced with MEM supplementedwith 10% FBS.

[0070] Luciferase expression was determined at 24 h with somemodification of a published

and magnesium free) and then treated with 0.5 ml of ice-cold lysisbuffer (25 mM Tris chloride pH 7.8, mM EDTA, 8 mM magnesium chloride, 1%Triton X-100, 1 mM DTT) for 10 min. The cell lysate mixture was scraped,transferred to 1.5 ml micro centrifuge tubes, and centrifuged for 7 minat 13,000 g at 4° C. to pellet debris.

[0071] Luciferase reporter gene expression was analyzed followingtransfection of HepG2 or COS 7 cells with peptide DNA condensatesprepared at stoichiometries ranging from 0.1-1.5 nmol of peptide per μgof DNA. A ten-fold enhancement in the gene expression level was achievedwhen chloroquine was included in the transfecting media. For eachpeptide condensing agent, the maximal reporter gene expression occurredat a peptide/DNA stoichiometry that corresponds to the fully condensedDNA as determined by the asymptote in the light scattering assay (FIG.1D, E, F). At stoichiometries greater than that required to achievecondensation the gene expression remained constant. Thereby, therelative gene expression levels were compared for each peptide DNAcondensate at a fixed stoichiometry of 0.8 or 1.0 nmol (DiCWK₃) ofpeptide per μg of DNA which was sufficiently for each peptide to fullycondense DNA.

[0072] Lysis buffer (300 μl), sodium-ATP (4 μl of a 180 mM solution, pH7, 4° C.) and cell lysate (100 μl, 4° C.) were combined in a test tube,briefly mixed and immediately placed in the luminometer. Luciferaserelative light units (RLU) were recorded on a Lumat LB 9501 (BertholdSystems, Germany) with 10 sec integration after automatic injection of100 μl of 0.5 mM D-luciferin (prepared fresh in lysis buffer withoutDTT). The RLU were converted into fmol using a standard curve generatedeach day using luciferase dissolved in Tris acetate pH 7.5 and stored at−20° C. The standard curve was constructed by adding a known amount ofthe enzyme (0.01-100 fmols with specific activity of 2.5 nU/fmol) to 35mm wells containing 40-70% confluent HepG2 or COS 7 cells. The cellswere processed as described above resulting in a standard curve with anaverage slope of 130,000 RLU per fmol of enzyme.

[0073] Protein concentrations were measured by Bradford assay usingbovine serum albumin as a standard. The amount of luciferase recoveredin each sample was normalized to milligrams of protein and the mean andstandard deviation obtained from each triplicate are

[0074] COS 7 cells were plated at 72,000 cells per well and grown to 50% confluency in DMEM (Gibco BRL) supplemented with penicillin (10,000U/ml), L-glutamine (200 mM),and 10% FBS for 24 hrs. The cells weretransfected as described for HepG2 cells.

[0075] Lipofectace™ (Gibco BRL, 1:2.5 w/w dimethyl dioctadecylammoniumbromide and dioleoyl phosphatidylethanolamine) was used to mediatenonspecific gene transfection according to the manufacturer'sinstructions. The ratio of DNA to lipofectace was optimized for both COS7 and HepG2 cells. An optimal DNA/lipofectace ratio was achieved bydissolving 10 μg of DNA in 100 μl of serum free media (SFM) followed byadding 60 μl of lipofectace prepared in 140 μl of SFM. The lipofectaceDNA complex was then diluted with 1.7 ml of SFM and used to transfectHepG2 or COS 7 cells for 5 hrs followed by replacing the transfectingmedia with supplemented 10% FBS. The cells were incubated for a total of24 hrs then harvested and analyzed for luciferase as described above.

[0076] Dose response curves were prepared by varying the dose from 1-50μg of DNA while keeping the peptide/DNA stoichiometry fixed at 0.6 nmolper μg of DNA and normalizing the volume to 0.5 ml. Alternatively, adose response curve for lipofectace was prepared by varying the DNA dosefrom 1-20 μg while keeping the stoichiometry of lipofectace to DNAconstant and normalizing the total volume of each dose to 2 ml with SFM.

[0077] Transfection of HepG2 with 10 μg of either uncomplexed DNA.AlkCWK_(3 or 8), DiCWK₃ or polylysine₁₉ DNA condensates failed toproduce a significant reporter gene expression (FIG. 3A). This resultsupported formulation experiments that predicted these peptides eitherfail to condense DNA (AlkCWK₃) or produced condensates that were large(0.7-3.1 μm). Alternatively, AlkCWK_(13 or 18) and DiCWK_(8, 13 or 18)DNA condensates each demonstrated significant gene transfer efficiencythat was two to three orders of magnitude greater than polylysine₁₉.Lipofectace mediated gene expression levels were also found to beidentical to peptide mediated expression levels in HepG2 cells (FIG.3A).

[0078] To verify that peptide mediated gene delivery was not dependenton the existence of cell type specific receptors, the reporter geneexpression in HepG2 cells was compared to COS 7 cells (FIG. 3B).Significant differences were observed for the transfection of COS 7

to produce measurable gene expression levels. AlkCWK₈, DiCWK₃ andpolylysine₉ DNA condensates each mediated a significant gene expressionin COS 7 cells despite their inactivity in transfecting HepG2 cells.However, the gene expression level mediated by these peptides was stillone to two orders of magnitude below that afforded by AlkCWK_(13 or 18)and DiCWK_(8, 13 and 18) (FIG. 3B). Also, lipofectace mediated geneexpression in COS 7 cells was one order of magnitude greater thanpeptide mediated gene delivery. These results suggest that the sizerestriction of peptide DNA condensates is less stringent in COS 7 cellscompared to HepG2 cells.

[0079] To establish the effect of dose response using peptide DNAcondensates, HepG2 cells were treated with escalating doses of AlkCWK₁₈DNA condensates and lipofectace DNA formulations. As demonstrated inFIG. 4, a dose response curve for the AlkCWK₁₈ DNA condensate plateausat 20 μg of DNA and remains constant at higher doses whereas thetoxicity of lipofectace appears above 10 μg of DNA (data not shown) andleads to reduced expression levels at higher doses.

[0080] From the above, it should be evident that the present inventionprovides compositions and methods that allow for efficient genedelivery. The development of homogenous peptides that actively condenseDNA into small particles is an important advance. Attachment of areceptor ligand such as a carbohydrate or peptide to a single cysteineresidue provides specificity to the gene delivery system. The abovemethods and compositions are amenable to use with virtually any gene ofinterest and permit the introduction of genetic material into a varietyof cells and tissues.

1 11 20 amino acids amino acid Not Relevant linear protein 1 Cys Trp LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys LysLys Lys 20 19 amino acids amino acid Not Relevant linear protein 2 LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15Lys Lys Lys 20 amino acids amino acid Not Relevant linear proteinBinding-site 1 /note= “The residue at this position is bound to anAlkaloid by a Sulfide.” 3 Cys Trp Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys 20 5 amino acids aminoacid Not Relevant linear protein Binding-site 1 /note= “The residue atthis position is bound to an Alkaloid by a Sulfide.” 4 Cys Trp Lys LysLys 1 5 10 amino acids amino acid Not Relevant linear proteinBinding-site 1 /note= “The residue at this position is bound to anAlkaloid by a Sulfide.” 5 Cys Trp Lys Lys Lys Lys Lys Lys Lys Lys 1 5 1015 amino acids amino acid Not Relevant linear protein Binding-site 1/note= “The residue at this position is bound to an Alkaloid by aSulfide.” 6 Cys Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys1 5 10 15 10 amino acids amino acid Not Relevant linear proteinBinding-site 5..6 /note= “The residues at these positions are joinedtogether by a Disulfide bond.” 7 Lys Lys Lys Trp Cys Cys Trp Lys Lys Lys1 5 10 20 amino acids amino acid Not Relevant linear proteinBinding-site 10..11 /note= “The residues at these positions are joinedtogether by a Disulfide bond.” 8 Lys Lys Lys Lys Lys Lys Lys Lys Trp CysCys Trp Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys 20 30 amino acidsamino acid Not Relevant linear protein Binding-site 15..16 /note= “Theresidues at these positions are joined together by a Disulfide bond.” 9Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Trp Cys Cys 1 5 1015 Trp Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 30 40amino acids amino acid Not Relevant linear protein Binding-site 20..21/note= “The residues at these positions are joined together by aDisulfide bond.” 10 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys 1 5 10 15 Lys Lys Trp Cys Cys Trp Lys Lys Lys Lys Lys LysLys Lys Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys Lys Lys 35 40 15 aminoacids amino acid Not Relevant linear protein 11 Cys Trp Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15

1. A method, comprising: a) providing: i) nucleic acid encoding one ormore gene products, ii) a tryptophan-containing peptide of less thanforty amino acids in length capable of binding to said nucleic acid, andiii) a cell having a cell membrane; b) binding saidtryptophan-containing peptide to said nucleic acid to make a complex; c)introducing said complex to said cell under conditions such that saidcomplex is delivered across said cell membrane.


3. The method of claim 2, wherein said peptide further comprisescysteine.
 4. The method of claim 3, wherein said peptide has thestructure Cys-Trp-(Lys)₁₈.
 5. The method of claim 4, wherein said Cys isalkylated.
 6. The method of claim 1, wherein said peptide is less thantwenty amino acids in length.
 7. A method of delivering nucleic acid tocells, comprising: a) providing: i) a cell having a receptor, ii) atarget binding moiety capable of binding to said receptor, iii) nucleicacid encoding one or more gene products, and iv) atryptophan-substituted polylysine of less than forty amino acids inlength capable of binding to said nucleic acid; b) conjugating saidtarget binding moiety to said tryptophan-substituted polylysine to forma carrier; c) coupling said carrier with said nucleic acid to form apharmaceutical composition; and d) introducing said pharmaceuticalcomposition to said cell under conditions such that said nucleic acid isdelivered to said cell.
 8. The method of claim 7, wherein said peptidefurther comprises cysteine.
 9. The method of claim 8, wherein saidpeptide has the structure Cys-Trp-(Lys)₁₈.
 10. The method of claim 9,wherein said Cys is alkylated.

length.
 12. A method of delivering nucleic acid to cells of an animal,comprising: a) providing: i) an animal containing cells having areceptor, ii) a target binding moiety capable of binding to saidreceptor, iii) nucleic acid encoding one or more gene products, and iv)a tryptophan-substituted polylysine of less than forty amino acids inlength capable of binding to said nucleic acid; b) conjugating saidtarget binding moiety to said tryptophan-substituted polylysine to forma carrier; c) coupling said carrier with said nucleic acid to form apharmaceutical composition; and d) administering said composition tosaid animal under conditions such that said nucleic acid is delivered tosaid cells.
 13. The method of claim 12, wherein said peptide furthercomprises cysteine.
 14. The method of claim 13, wherein said peptide hasthe structure Cys-Trp-(Lys)₁₈.
 15. The method of claim 14, wherein saidCys is alkylated.
 16. The method of claim 12, wherein said peptide isless than twenty amino acids in length.
 17. A tryptophan-containingpeptide of less than forty amino acids in length capable of binding tonucleic acid.
 18. The peptide of claim 17, wherein said peptide furthercomprises lysine.
 19. The peptide of claim 18, wherein said peptidefurther comprises cysteine.
 20. The peptide of claim 19, wherein saidpeptide has the structure Cys-Trp-(Lys)₁₈.
 21. The peptide of claim 20,wherein said Cys is alkylated.
 22. The peptide of claim 17, wherein saidpeptide is less than twenty amino acids in length.